This application describes plans to develop new potentiometric fluorescent membrane probes, new methods for using them and the application of this methodology to several important problems in cell biology. In addition to one new chromophore base on a predicted electrochromic mechanism, the synthesis of amphipathic cation complexing dyes which would detect cation concentrations at the membrane surface is planned. A combination of molecular mechanics and pi-molecular orbital theory will be used to design trinuclear dyes which will undergo chromophore interchanges via potential- dependent conformational changes. A complete database including molecular structures will be developed for the 1400 dyes screened over the past 15 years in several laboratories; this will permit the use of structure/activity algorithms to extract structural attributes which impart sensitivity to dyes. New methods which will be further developed include dual wavelength ratiometric measurements of potential with membrane staining dyes and the measurement of membrane potential of individual mitochondria in living cells via confocal microscopy. The effect of intracellular calcium and pH on mitochondrial potential will be studied via combinations of the appropriate fluorescent indicators. Inhibitors of heme synthesis in reticulocytes will be used to probe for the possible interrelationships between mitochondrial activity, intracellular calcium and the feedback mechanism for controlling receptor mediated iron transport. A fast membrane staining dye will be used to label targets for cytotoxic T lymphocytes in order to correlate cell physiological events in the effector cell with the delivery of the lethal hit. The ratiometric approach will be used to probe for both resting and stimulated membrane potential changes during the cell cycle of HeLa cells.